Objective:To construct and prepare the recombinant adenovirus vector co-expressing the axonal attractant netrin-1 and report gene EGFP as preparation for later use for genetic transfection. Methods:The netrin-1 was cloned by RT-PCR and the then subcloned into shuttle vector pDC316-CMV which carries the report gene EGFP. Subsequently, this newly constructed plasmid pDC316-netrin, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGlox_E1.3Cre. Based on homologous recombination of two plasmids within HEK293 cells, the recombinant adenovirus vector carrying netrin-1, Ad5-netrin-CMV-EGFP, was created. Ad5-netrin-CMV-EGFP was subsequently identified by PCR, purified using repeated plaque passages, proliferated using freezing and melting with HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus carrying rat netrin-1 was confirmed by PCR, and its titration value determined by TCID50 assay was 5.2 × 109pfu/ml. The positivity rate of adenovirus infected cells was over 40%. Conclusion:The recombinant adenovirus carrying rat netrin-1 was successfully constructed. The newly constructed can produce sufficiently high titration value with HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.